Diapedesis is a critical step in the inflammatory response in which leukocytes migrate across endothelial cells out of the circulation and into inflamed tissues. We have found that the molecule PECAM-1, expressed at the borders of endothelial cells and on leukocytes, is a critical regulator of diapedesis. Interfering with its function can block 80- 90% of monocyte and neutrophil migration across the endothelium in vitro and in vivo in several models of inflammation. We know how to interfere with PECAM by blocking critical domains on its external surface, but we do not know how it functions to regulate diapedesis. We recently identified an internal pool of PECAM-bearing membrane that moves to surround transmigrating leukocytes ("targeted recycling") during diapedesis. This action was required for diapedesis to proceed. The aims of this application are to investigate how endothelial cells regulate diapedesis with special attention to the role of PECAM therein: 1. An increase in cytosolic free calcium ion concentration is required for diapedesis to proceed. We will investigate whether PECAM plays a role in triggering this rise, and whether the calcium rise is necessary to trigger or sustain targeted recycling of PECAM-bearing membrane. 2. Phosphorylation of tyrosine residues on the cytoplasmic tail of PECAM mediates signaling in many systems. We will investigate whether such phosphorylation takes place during diapedesis of leukocytes, whether it is a cause or effect of diapedesis, and whether it is required for targeted recycling. 3. The interrelationships of the triggered rise in cytosolic free calcium, PECAM phosphorylation, and targeted recycling will be explored to determine if one regulates the other, or if they function independently to promote diapedesis. We will determine whether other molecules in the junction are involved in the targeted recycling and whether they can take over for PECAM if it is blocked. 4. Interfering with PECAM function blocks inflammation in vivo, but has only been tested in acute inflammatory models. We will test the role of PECAM in murine models of three chronic inflammatory diseases: rheumatoid arthritis, atherosclerosis, and multiple sclerosis. For these studies we will use mice rendered genetically deficient in PECAM and transgenic mice that constitutively secrete a circulating competitive inhibitor of PECAM. These studies will show whether PECAM represents a reasonable therapeutic target for chronic anti-inflammatory therapy. [unreadable] [unreadable]